Insertional mutagens have been particularly valuable for geneticists. In the mouse, the use of insertional mutagenesis for germline genetic screens has been limited to transgene insertion and gene trapping in embryonic stem cell lines. In this proposal, we further characterize and develop a method for in vivo gene trapping in the mouse using a transposable element. The method relies on the use of a synthetic transposable element called Sleeping Beauty (SB), which is a reconstructed member of the Tcl/mariner family of DNA transposable elements. We have demonstrated that efficient mobilization of SB transposons can be made to occur in the germline of transgenic mice and that novel transposon insertions can occur in or near genes. We propose optimization of the SB system for germline and somatic cell insertional mutagenesis in the mouse. Our goals include additional improvements in the transposon vector and the transposase enzyme, some of which have already been made and are now being tested mice, and some of which will be achieved using a novel selection method that utilizes the retroviral life cycle. We will determine how the site of transposon excision is repaired in the presence of a perfect copy of the transposon on a homologous chromosome and on a genetic background which is defective for non-homologous end joining. We will also create a system in which potentially oncogeneic transposons are mobilized in somatic cells with an aim to create models of cancer in the mouse caused by transposon insertional mutagenesis. Finally, we have shown that the transposable element can be used for stable long-term gene transfer into somatic cells of mice and we propose to use this technique to create models of cancer in the mouse in which oncogenes and putative oncogenes are directly transferred into somatic target cells. In summary, this proposal aims to utilize, further improve, and better understand a mammalian active transposable element for use in mouse germline genetic screens, somatic cell mutagenesis for cancer genes, and as a gene transfer vehicle for cancer studies.